Atrazine-induced degranulation of thyroid mast cells in peripubertal and adult rats

Atrazine is a commonly used pesticide in the US and the non-EU countries. It is classified as an endocrine-disrupting chemical and is well-known for its reproductive toxicity in mammals and lower vertebrates. The study on atrazine effects on thyroid mast cells was performed on juvenile/peripubertal and adult male Wistar rats orally gavaged with atrazine at doses of 50 mg/kg of body weight (bw) or 200 mg/kg bw. In order to visualize the mast cell population within the thyroid gland, a histochemical staining method of toluidine blue was used. The results of the histological evaluation demonstrated a prominent increase in mast cell degranulation in both age groups and at both atrazine doses. According to the stereological analysis, a statistically significant decrease in the mast cell volume density in the young rats exposed to a higher dose of atrazine was found when compared to the corresponding control. The numerical density of mast cells significantly decreased in a higher-dose atrazine treated adults in comparison to the control. The obtained data suggest that atrazine-affected mast cells would probably have a consequent influence on thyroid follicular cells and/or thyroid microvasculature via paracrine action of released mediators, but might also be involved in already suggested thyroid cancerogenesis.     

Atrazine,desethylatrazine (DEA) and desisopropylatrazine (DIA) degradation by Pleurotus ostreatus INCQS 40310

Abstract

Atrazine is the most common herbicide applied in crops of economic relevance, such as sugar cane, soybean, and corn. Atrazine and its derivatives desethylatrazine (DEA) and desisopropylatrazine (DIA) are toxic to the environment, affecting animal and human health. Thus, this study aimed to evaluate the degradation of atrazine and its derivatives by the fungus Pleurotus ostreatus INCQS 40310, as well as the potential of the enzymes involved in this process. P. ostreatus INCQS 40310 was able to degrade atrazine (82%), DEA (71%), and DIA (56%) over 22?days of fungal cultivation. Proteomic analysis indicated the participation of hydrolases and peroxidases during the degradation process. Additionally, resting cells of the fungus were tested to verify the action of intracellular enzymes in the degradation process, suggesting the participation of cytochrome P450 enzymatic complex. Resting cells experiments promoted the degradation of 50% of atrazine, 36% of DIA, 30% of DEA. So far, this is the first work evaluating the biodegradation of DEA and DIA by fungus.     

The presence of atrazine and atrazine-degrading bacteria in the residential, cattle farming, forested and golf course regions of Lake Oconee

Aims: To assess the concentration of atrazine in Lake Oconee and develop a qPCR assay as a potential marker for the presence of atrazine‐degrading bacteria indicating atrazine contamination. Methods and Results: Water and sediment samples were collected from the Oconee Lake at four golf course sites, two residential sites, one cattle farming site and a forested site. Atrazine concentration at the study sites was determined using an ELISA kit and indicated the presence of atrazine from 0·72 ppb at the forested sites to 1·84 ppb at the golf course sites. QPCR results indicate the presence of atzA gene (atrazine chlorohydrolase) from 1·51 × 10² gene copies at the residential sites to 3·31 × 10⁵ gene copies per 100 ml of water at the golf course regions of the lake and correlated (r = 0·64) with atrazine concentration. Sediment samples had higher atzA gene copies compared with the water samples (P < 0·05). Conclusions: Atrazine concentration and the highest quantity of atzA gene were detected in the golf course regions of the lake. Overall, atrazine concentration monitored in Lake Oconee was below the Environment Protection Agency (EPA) regulatory standards. Significance and Impact of the Study: Quantitative PCR is an efficient technique for assessing the presence of atrazine catabolism gene as a functional marker for atrazine‐degrading bacteria and the presence of atrazine contamination.     

Consumption of Triazine Herbicide Atrazine by Laccase-positive and Laccase-negative Strains of Soil Fungus Mycelia sterilia INBI 2-26

Asporogenic fungus Mycelia sterilia INBI 2-26 isolated from tropical soils with high residual dioxin content (as a result of Agent Orange defoliant treatment during the Vietnamese–American war) and capable of atrazine decomposition was treated to obtain protoplasts. This technique resulted in isolation of laccase-positive and laccase-negative clones. Atrazine consumption by liquid surface cultures of Mycelia sterilia INBI 2-26 was monitored by using enzyme immune assay and reversed-phase HPLC. Atrazine (20 g/ml) stimulated fungal growth. The laccase-positive clone consumed up to 80% of atrazine within four weeks. However, no correlation of atrazine consumption and laccase activity in the culture medium was observed. Moreover, the laccase-negative clone was also capable of consuming at least 60–70% of atrazine within three weeks. Surprisingly, in the corresponding control set (cultivation of laccase-negative clone without atrazine) an unidentified metabolite having a retention time and UV-spectrum similar to those of atrazine was also found. It was concluded that the presence of laccase was not a crucial factor in atrazine consumption by this fungus.     

Atrazine induced changes in elemental and biochemical composition and nitrate reductase activity in Chlamydomonas reinhardtii

Herbicides play an important role in agricultural practices but the introduction of these compounds into the aquatic environment can have severe consequences for non-target organisms such as microalgae. The ubiquitous green freshwater microalga Chlamydomonas reinhardtii, a model species in all aspects of microalgal physiology, was used to assess the toxicity of atrazine, one of the most widely used herbicides throughout the world. Atrazine acts on photosynthesis and therefore can affect non-target primary producers, such as microalgae.

Growth, dry weight, elemental composition, photosynthetic pigments and protein contents and nitrate reductase activity were studied. After 96 h of exposure to different atrazine concentrations all the parameters studied were affected, but different sensitivities to the herbicide were shown. Nitrate reductase (NR) activity was strongly affected even at an atrazine concentration that did not affect growth (0.1 µM); the lowest concentrations of atrazine assayed (0.1 and 0.25 µM) provoked a > 40% decrease in NR activity and NR decreased > 80% with atrazine concentrations of 0.5 µM. C/N ratio was also affected by all the atrazine concentrations assayed. Nitrate reductase activity and C/N ratio were better indicators of the cellular stress state than data on other biochemical components or growth rate. Among cell parameters assayed, the NR activity stood out as a sensitive cytotoxicity endpoint and the activity of this enzyme can be suggested as a sensitive biomarker of stress induced by atrazine in C. reinhardtii.     

Selection of atrazine tolerant soybean calli and expression of that tolerance in regenerated plants

Lines of soybean [Glycine max (L.)] tolerant of atrazine were developed by an in vitro and in vivo atrazine challenge. Cotyledonary node plus epicotyl explants from mature germinated seed of soybean introduction PI 438489B were cultured on RV-5 medium containing 48 mg active ingredient (a.i.)/l atrazine for one month. Most of the explants (66%) on medium containing atrazine, and 10% on medium without atrazine died. Explants surviving exposure to atrazine callused and organogenically regenerated shoots developed. Soil around R₀ plants regenerated from atrazine tolerant shoots and nonatrazine challenged shoots (controls) were subsequently tested in vivo for atrazine tolerance. All controls died. Seeds were collected from atrazine tolerant R₀ plants. Two weeks after planting, emerged R₁ seedlings were tested in vivo for atrazine tolerance as the R₀ plants were. This procedure was repeated on the R₂ plants. All nonatrazine selected control plants died when exposed to this herbicide. Atrazine tolerant R₂ plants were maintained in atrazine amended soil and appeared as healthy and vigorous as the control growing in atrazine free soil.Missouri Agricultural Experiment Station, University of Missouri, Columbia, Missouri. Journal Series Number 11.340Mention of tradenames does not constitute a guarantee or warranty of the product by the University of Missouri and does not imply their approval to the exclusion of other products.     

D2O effects on association behavior and binding properties of phospho- and dephospho-cytoplasmic malic dehydrogenase

Changes in the concentration of three major carbohydrates, e.g., glycogen, trehalose, and cellulose, were determined during differentiation of Dictyostelium discoideum in a stage study. These three carbohydrates consituted 50–63% of the total carbohydrates. Total carbohydrate content per cell aliquot did not change between the aggregation and sorocarp stages of differentiation. The isolation, purification, and characterization of cellulose is described. Cellulose consisted of an alkali-insoluble (alpha) and an alkali-soluble (beta) fraction. Total cellulose accumulated from very low amounts in late pseudoplasmodium cells to about 35% of carbohydrates in mature sorocarps at a rate of 0.07 μmole glucose equiv/min/ml packed cell volume. Purified alkali-insoluble cellulose constituted about 19% of total carbohydrates in mature sorocarps and accumulated at a rate of 0.035 μmole glucose equiv/min/ml packed cell volume. Trehalose constituted 10–11% of the carbohydrates in sorocarps and accumulated at a rate of 0.035 μmole glucose equiv/min/ml packed cell volume. Glycogen, comparing several methods of determination, was rapidly degraded between the culmination and sorocarp stages of differentiation at an average rate of 0.05 μmole glucose equiv/min/ml packed cell volume. The major portion of glycogen was soluble in TCA and constituted 35% of total carbohydrates in aggregated cells and about 11% in mature sorocarps. A minor fraction of glycogen, about 15% of total carbohydrates in aggregated cells, was solubilized by KOH from a TCA precipitate. A mild acidic treatment of solubilized cell constituents increased the glycogen content by 55%, as judged by an enzymatic assay.     

Some life history responses of the cladoceran Ceriodaphnia cf. dubia to variations in population density at two different food concentrations

Atrazine is the most commonly used agricultural herbicide in the midwestern cornbelt of the U.S.A. This project investigated atrazine bioconcentration in Cladophora glomerata (L.) Kütz, the dominant algae by biomass in nutrient enriched rivers. C. glomerata concentrates many organic and inorganic materials from solution, and by considering the partitioning coefficient of atrazine (Log K _ow=2.56), the uptake of atrazine by this green algae was expected. The bioconcentration factor of C. glomerata ranged from 29 to 5223 over a one year sampling period, and bioconcentration was found to be a function of maximum biomass increase (due to high nutrients) and discharge. Uptake increased at low discharges as a result of increased contact time between cells and water. We found that C. glomerata was able to bioconcentrate a yearly average of 54% of the available atrazine from the water. Efficiency of uptake increased during the summer growing season of the algae (81%) and was 34% during the winter months. This field study demonstrates the ability of C. glomerata to bioconcentrate atrazine to a high degree and suggests an alternative and quicker biological removal of atrazine from agriculturally polluted rivers.     

Growth of Halodule wrightii in culture and the effects of cropping,light, salinity and atrazine

The interaction of three environmental variables, light, salinity and cropping, with the effects of the herbicide atrazine on Halodule wrightii Ascherson was investigated in the laboratory. Atrazine at 30 ppm caused a significant reduction in survival of ramets, production of new ramets, above-ground biomass and growth, when compared to Halodule wrightii not exposed to atrazine. The three levels of each environmental condition did not alter the toxicity of atrazine to Halodule wrightii.     

Isoenzyme Patterns of Solanum Nigrum and the Cybrid Plant containing S. Nigrum Genome and S. Tuberosum Plastome

Transfer of chloroplasts from Solanum tuberosum into S. nigrum cell resulted in an atrazine sensitive cybrid plant. The shoots of this cybrid were bleached under atrazine stress. The cybrid displayed identical isoenzyme patterns that have been found in S. nigrum, and thus nuclei of the cybrid plant cells did not integrate any chromosomes or chromosome fragments from S. tuberosum nuclei. Under atrazine stress, differences in the isoenzyme expression were found in both the cybrid and original plants. In the peroxidase patterns, POX-4 was detected while POX-1 disappeared. Esterase patterns were less influenced, EST-4 was expressed in both plants under the influence of atrazine. Atrazine treatment of both cybrid and original plant shoots had specific effects on carbonic anhydrase, malate dehydrogenase, and glutamate oxaloacetate transaminase. The number and/or the staining intensity of bands corresponding to these isoenzymes decreased under atrazine stress more in cybrid plants (atrazine sensitive) than in S. nigrum (atrazine resistant).     

Combined Effects of Atrazine and Chlorpyrifos on Susceptibility of the Tiger Salamander to Ambystoma tigrinum Virus

Several hypotheses have been examined as potential causes of global amphibian declines, including emerging infectious diseases and environmental contaminants. Although these factors are typically studied separately, animals are generally exposed to both stressors simultaneously. We examined the effects of the herbicide atrazine and the insecticide chlorpyrifos on the susceptibility of tiger salamander larvae, Ambystoma tigrinum, to a viral pathogen, Ambystoma tigrinum virus (ATV). Environmentally relevant concentrations of atrazine (0, 20, 200 μg/L) and chlorpyrifos (0, 2, 20, 200 μg/L) were used along with ATV in a fully factorial experimental design whereby individually housed, 4-week-old larvae were exposed for 2 weeks. Atrazine alone was not lethal to larvae, and chlorpyrifos alone was lethal only at the highest concentration. When combined with ATV, chlorpyrifos increased susceptibility to viral infection and resulted in increased larval mortality. A significant interactive effect between atrazine and ATV was detected. Atrazine treatments slightly decreased survival in virus-exposed treatments, yet slightly increased survival in the virus-free treatments. These findings corroborate earlier research on the impacts of atrazine, in particular, on disease susceptibility, but exhibit greater effects (i.e., reduced survival) when younger larvae were examined. This study is the first of its kind to demonstrate decreases in amphibian survival with the combination of pesticide and a viral disease. Further examination of these multiple stressors can provide key insights into potential significance of environmental cofactors, such as pesticides, in disease dynamics.     

Encapsulation of Pseudomonas sp. ADP cells in electrospun microtubes for atrazine bioremediation

Electrospun hollow polymeric microfibers (microtubes) were evaluated as an encapsulation method for the atrazine degrading bacterium Pseudomonas sp. ADP. Pseudomonas sp. ADP cells were successfully incorporated in a formulation containing a core solution of polyethylene oxide dissolved in water and spun with an outer shell solution made of polycaprolactone and polyethylene glycol dissolved in a chloroform and dimethylformamide. The resulting microtubes, collected as mats, were partially collapsed with a ribbon-like structure. Following encapsulation, the atrazine degradation rate was low (0.03?±?0.01?mg atrazine/h/g fiber) indicating that the electrospinning process negatively affected cell activity. Atrazine degradation was restored to 0.5?±?0.1?mg atrazine/h/g fiber by subjecting the microtubes to a period of growth. After 3 and 7?days growth periods, encapsulated cells were able to remove 20.6?±?3 and 47.6?±?5.9?mg atrazine/g mat, respectively, in successive batches under non-growth conditions (with no additional electron donor) until atrazine was detected in the medium. The loss of atrazine degrading capacity was regained following an additional cell-growth period.     

Cumulative effect of low and high atrazine concentrations on <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis> plants

Atrazine belongs to the widely used herbicides blocking the electron transport chain in chloroplasts, thus resulting in the generation of active oxygen species. In the present work, we demonstrated that, at low concentrations mimicking residual amounts, atrazine enhanced the susceptibility of Arabidopsis plants to further treatments with the same herbicide applied at the recommended field rate. Arabidopsis thaliana plants were treated three times (at five-day intervals) with 1 µM atrazine. Five days after the last treatment, the plants were sprayed with 5 mM atrazine. Atrazine increased the levels of lipid peroxidation products, hydrogen peroxide, and ion leakage, and caused changes in the activities of antioxidant enzymes, such as superoxide dismutase, guaiacol peroxidase, and catalase.From Fiziologiya Rastenii, Vol. 52, No. 2, 2005, pp. 243–249.Original English Text Copyright © 2005 by Ivanov, Alexieva, Karanov.This article was submitted by the authors in English.This revised version was published online in April 2005 with a corrected cover date.     

Transgenic tobacco plants expressing atzA exhibit resistance and strong ability to degrade atrazine

Atrazine chlorohydrolase (AtzA) catalyzes hydrolytic dechlorination and can be used in detoxification of atrazine, a herbicide widely employed in the control of broadleaf weeds. In this study, to investigate the potential use of transgenic tobacco plants for phytoremediation of atrazine, atzA genes from Pseudomonas sp. strain ADP and Arthrobacter strain AD1 were transferred into tobacco. Three and four transgenic lines, expressing atzA-ADP and atzA-AD1, respectively, were produced by Agrobacterium-mediated transformation. Molecular characterization including PCR, RT-PCR and Southern blot revealed that atzA was inserted into the tobacco genome and stably inherited by and expressed in the progenies. Seeds of the T₁ transgenic lines had a higher germination percentage and longer roots than the untransformed plants in the presence of 40–150 mg/l atrazine. The T₂ transgenic lines grew taller, gained more dry biomass, and had higher total chlorophyll content than the untransformed plants after growing in soil containing 1 or 2 mg/kg atrazine for 90 days. No atrazine residue remained in the soil in which the T₂ transgenic lines were grown (except 401), while, in the case of the untransformed plants, 0.91 mg (81.3%) and 1.66 mg (74.1%) of the atrazine still remained in the soil containing 1 and 2 mg/kg of atrazine, respectively, indicating that the transgenic lines could degrade atrazine effectively. The transgenic tobacco lines developed could be useful for phytoremediation of atrazine-contaminated soil and water.     

Growth and production characteristics of permeabilized Coleus blumei cells in immobilized fed-batch culture

Summary Permeabilized Coleus blumei cells were cultivated in an immobilized state to study the effect of dimethyl sulfoxide (DMSO) concentrations and growth regulators on cell growth and rosmarinic acid (RA) production characteristics. Luffa (the fibrous skeleton of mature fruit of Luffa cylindrica) was a good support matrix for cell immobilization because of its high void volume. Maximum cell loading capacity was 1.33 g dry cell weight (DCW)/g dry Luffa. The experiments were done in shake flasks with no free medium. The medium was supplied in a fed-batch mode to avoid the flotation of Luffa pieces. The sucrose in the medium was completely hydrolyzed to glucose and fructose without any sugar accumulation in the medium. The cell viability was slightly higher in the cells on top of the Luffa than those in the middle. Cell growth rate and rosmarinic acid (RA) production were approximately half that obtained in cell suspension cultures. Cell yield (g DCW/g glucose) was similar to that of cell suspension cultures. The absence of growth regulators did not promote an increase of RA production but did decrease the cell mass. The second step preconditioning with 0.5% DMSO did not improve the cell's adaptability to higher DMSO concentrations and the cell mass did not increase with 2.5% DMSO.     

Characterization of S-Triazine Herbicide Metabolism by a Nocardioides sp. Isolated from Agricultural Soils

Atrazine, a herbicide widely used in corn production, is a frequently detected groundwater contaminant. Nine gram-positive bacterial strains able to use this herbicide as a sole source of nitrogen were isolated from four farms in central Canada. The strains were divided into two groups based on repetitive extragenic palindromic (rep)-PCR genomic fingerprinting with ERIC and BOXA1R primers. Based on 16S ribosomal DNA sequence analysis, both groups were identified as Nocardioides sp. strains. None of the isolates mineralized [ring-U-¹⁴C]atrazine. There was no hybridization to genomic DNA from these strains using atzABC cloned from Pseudomonas sp. strain ADP or trzA cloned from Rhodococcus corallinus. S-Triazine degradation was studied in detail in Nocardioides sp. strain C190. Oxygen was not required for atrazine degradation by whole cells or cell extracts. Based on high-pressure liquid chromatography and mass spectrometric analyses of products formed from atrazine in incubations of whole cells with H₂¹⁸O, sequential hydrolytic reactions converted atrazine to hydroxyatrazine and then to the end product N-ethylammelide. Isopropylamine, the putative product of the second hydrolytic reaction, supported growth as the sole carbon and nitrogen source. The triazine hydrolase from strain C190 was isolated and purified and found to have a K_m for atrazine of 25 μM and a V_max of 31 μmol/min/mg of protein. The subunit molecular mass of the protein was 52 kDa. Atrazine hydrolysis was not inhibited by 500 μM EDTA but was inhibited by 100 μM Mg, Cu, Co, or Zn. Whole cells and purified triazine hydrolase converted a range of chlorine or methylthio-substituted herbicides to the corresponding hydroxy derivatives. In summary, an atrazine-metabolizing Nocardioides sp. widely distributed in agricultural soils degrades a range of s-triazine herbicides by means of a novel s-triazine hydrolase.     

Use of atrazine sensitive leguminous plants as biological indicators to evaluate the atrazine degradation efficiency of a bacterial inoculum

Atrazine sensitive leguminous plants were grown in a soil spiked with atrazine and augmented with an atrazine-degrading bacterium, Arthrobacter sp. strain MCM B-436, to ascertain its degradative efficiency. Germination and survival of plants was correlated with atrazine removal from soil. This experiment was carried out at laboratory as well as field level, showing consistent results. This bioindicator approach serves as an efficient measure for atrazine removal and could be easily adapted to determine atrazine degradation efficiency of other microbial strains.     

Reduction of Nitrate and Nitrite in Lambsquarters (Chenopodium album) Biotypes Resistant and Susceptible to Atrazine Toxicity

The nitrite-reducing activity of the normal susceptible biotype of lambsquarters (Chenopodium album L.) was strongly inhibited by atrazine in the assay medium, both in the case of the in vivo assays of leaf discs in light, and in vitro photoreduction assays of crude extracts. In vitro assays of crude extracts with methylviologen or ferredoxin supplying the reducing potential were not inhibited by atrazine. In the resistant biotype, inhibition of nitrite reduction did not occur with any of the above assays. Thus, it appears that atrazine does not inhibit nitrite reductase itself, but rather the availability of photosynthetically supplied electrons for the reduction. Atrazine had no effect when added to the media for either in vivo or in vitro assays of nitrate reduction by either the susceptible or resistant biotype.     

4-Amino-2-chloro-1,3,5-triazine: A New Metabolite of Atrazine by a Soil Bacterium

The degradation of herbicide atrazine (2-chloro-4-ethylamino-6-isopropylamino-1, 3, 5-triazine) by a soil bacterium is reported. The bacterium involved is a species of Nocardia, which utilizes the atrazine as the sole source of carbon and nitrogen. A new metabolite, 4-amino-2-chloro-1, 3, 5-triazine, of the degradation of atrazine in the presence of glucose has been identified. The results further substantiated that atrazine can be degraded by soil microorganisms and indicated that deamination can also occur, as well as dealkylation. 4-Amino-2-chloro-1,3,5-triazine did not show phytotoxic activity to oat (Avena sativa L.), demonstrating that deamination insures detoxification.